UC San Diego

Nuclear factor-κB (NF-κB) is an inducible transcription factor involved in many cellular processes, such as inflammation, innate immune responses, and cell survival. The inhibitor of NF-κB Kinase (IKK) complex is central to the induction and nuclear translocation of NF-κB. Activation of IKK catalytic activity in response to TNF-α and other canonical inducers of NF-κB requires formation of non-degradative linear polyubiquitin chains and their association with its NEMO subunit. NEMO, or NF-κB essential modulator, is a component of the IKK complex that is required for canonical activation of NF-κB. Without NEMO, the canonical pathway is disrupted and NF-κB cannot be induced to translocate into the nucleus. Past observations suggested that the IKK NEMO subunit, upon noncovalent association with linear polyubiquitin chains, mediates a second protein-protein interaction with the catalytic IKK2 subunit and “primes” the complex for kinase catalytic activity via activation loop phosphorylation. The purpose of this dissertation is to investigate the direct involvement of NEMO in promoting catalytically active IKK and NF-κB-mediated gene expression in response to TNF-α and to establish cell-based systems through which novel NEMO-dependent mechanisms of IKK regulation can be identified. To test the involvement of NEMO in canonical NF-κB signaling, mutations that disrupt the protein-protein interactions of NEMO can be introduced in HEK293T using CRISPR-Cas9 prime editing and the resulting mutant cell lines can then be used against various canonical inducers. We successfully generated two mutant NEMO cell lines: one engineered to disrupt the secondary binding site and the other to disrupt the linear ubiquitin binding site. Both mutations displayed varying degrees of decrease in TNF-α-induced NF-κB activation as evidenced by IKK2 phosphorylation, IκBα degradation, RelA accumulation in the nucleus, and target gene expression levels. Luciferase reporter assays further confirmed that disruption of the secondary binding site in NEMO resulted in significantly decreased levels of TNF-α-dependent NF-κB activation, though not to the same extent as cells mutated to disrupt the noncovalent interaction of NEMO with linear ubiquitin. Future studies will incorporate the validated NEMO mutations into another cell line capable of responding to canonical NF-κB inducers and further examine NEMO’s involvement in canonical pathway activation.